The present invention relates to variants of cyclomaltodextrin glucanotransferase of increased product specificity.
Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), also designated cyclodextrin glucanotransferase or cyclodextrin glycosyltransferase, in the following termed CGTase, catalyses the conversion of starch and similar substrates into cyclomaltodextrins via an intramolecular transglycosylation reaction, thereby forming cyclomaltodextrins, in the following termed cyclodextrins (or CD), of various sizes. Commercially most important are cyclodextrins of 6, 7 and 8 glucose units, which are termed xcex1-, xcex2-, and xcex3-cyclodextrins, respectively. Commercially less important are cyclodextrins of 9, 10, and 11 glucose units, which are termed xcex4-, xcex5-, and xcex6-cyclodextrins, respectively.
Cyclodextrins are thus cyclic glucose oligomers with a hydrophobic internal cavity. They are able to form inclusion complexes with many small hydrophobic molecules in aqueous solutions, resulting in changes in physical properties, e.g. increased solubility and stability and decreased chemical reactivity and volatility. Cyclodextrins find applications particularly in the food, cosmetic, chemical and pharmaceutical industries.
Most CGTases have both starch-degrading activity and transglycosylation activity. Although some CGTases produce mainly xcex1-cyclodextrins and some CGTases produce mainly xcex2-cyclodextrins, CGTases usually form a mixture of xcex1-, xcex2- and xcex3-cyclodextrins. Selective precipitation steps with organic solvents may be used for the isolation of separate xcex1-, xcex2- and xcex3-cyclodextrins. To avoid expensive and environmentally harmful procedures, the availability of CGTases capable of producing an increased ratio of one particular type of cyclodextrin, in particular with respect to xcex1-, xcex2- or xcex3-cyclodextrin, is desirable.
WO 96/33267 (Novo Nordisk) describes CGTase variants showing a modified substrate binding and/or product selectivity. Although CGTase variants produced by mutation at positions 47, 145, 146, 147, 196 or 371 have been described, the specific CGTase variants of this invention have never been described or even suggested.
The present invention provided novel CGTase variants of increased product specificity. Although CGTase variants of increased product specificity have been described in the prior art (WO 96/33267), the CGTase variants of the present invention have never been described or suggested.
Among the tremendous number of possible CGTase variants we have now succeeded in finding a limited number of variants showing increased product specificity when compared to the wild-type enzyme.
Accordingly the invention provides a CGTase variant of increased product specificity, in which one or more of the amino acid residues corresponding to the following positions have been introduced by substitution and/or insertion (CGTase Numbering):
(i) Position 47: 47C; 47D; 47E; 47F; 47G; 47I; 47K; 47N; 47P; 47R;
47S; 47T; 47V; 47W; or 47Y;
(ii) Position 145: 145D; 145H; 145I; 145N; 145Q; or 145V;
(iii) Position 146: 146H, 146K; 146L; 146T; 146V; or 146Y;
(iv) Position 147: 147C; 147D; 147E; 147N; 147Q;
(v) Position 196: 196C; 196E; 196F; 196G; 196H; 196I; 196K; 196L; 196M; 196P; 196Q; 196R; 196T; 196V; or 196W; 196Y and/or
(vi) Position 371: 371C; 371E; 371F; 371H; 371I; 371K; 371L; 371M; 371Q; 371R; 371T; 371V; or 371W.
Amino Acids
In the context of this invention the following symbols and abbreviations for amino acids and amino acid residues are used:
A=Ala=Alanine
C=Cys=Cysteine
D=Asp=Aspartic acid
E=Glu=Glutamic acid
F=Phe=Phenylalanine
G=Gly=Glycine
H=His=Histidine
I=Ile=Isoleucine
K=Lys=Lysine
L=Leu=Leucine
M=Met=Methionine
N=Asn=Asparagine
P=Pro=Proline
Q=Gln=Glutamine
R=Arg=Arginine
S=Ser=Serine
T=Thr=Threonine
V=Val=Valine
w=Trp=Tryptophan
Y=Tyr=Tyrosine
B=Asx=Asp or Asn
Z=Glx=Glu or Gln
X=Xaa=Any amino acid
*=Deletion or absent amino acid
CGTase Variants
A CGTase variant of this invention is a CGTase variant or mutated CGTase, having an amino acid sequence not found in nature.
A CGTase variant or mutated CGTase of this invention may be considered a functional derivative of a precursor CGTase enzyme (i.e. the native, parental, or wild-type enzyme), and may be obtained by alteration of a DNA nucleotide sequence of a precursor gene or its derivatives, encoding the precursor enzyme. The CGTase variant or mutated CGTase may be expressed and produced when the DNA nucleotide sequence encoding the CGTase variant is inserted into a suitable vector in a suitable host organism. The host organism is not necessarily identical to the organism from which the precursor gene originated.
In the literature, enzyme variants have also been referred to as mutants or muteins.
CGTase Numbering
In the context of this invention a specific numbering of amino acid residue positions in CGTase enzymes is employed. By alignment of the amino acid sequences of various known CGTases it is possible to unambiguously allot a CGTase amino acid position number to any amino acid residue position in any CGTase enzyme, which amino acid sequence is known.
Using the numbering system originating from the amino acid sequence of the CGTase obtained from Bacillus circulans Strain 251, aligned with the amino acid sequence of a number of other known CGTases, it is possible to indicate the position of an amino acid residue in a CGTase enzyme unambiguously.
This CGTase Numbering system has been described in WO 96/33267, and its equivalent, U.S. Pat. No. 6,004,790, see Table 1, pages 9-31 (in which table Bacillus circulans Strain 251 is represented as a). Table 1 of WO 96/33267 and its equivalent U.S. Pat. No. 6,004,790 also show the protein sequences of a number of relevant CGTase and is hereby incorporated by reference.
In describing the various CGTase variants produced or contemplated according to the invention, the following nomenclatures are adapted for ease of reference:
[original amino acid; Position; Substituted amino acid]
Accordingly, the substitution of serine with alanine in position 145 is designated as S145A.
Amino acid residues which represent insertions in relation to the amino acid sequence of the CGTase from Bacillus circulans Strain 251, are numbered by the addition of letters in alphabetical order to the preceding CGTase number, such as e.g. position 91aF for the xe2x80x9cinsertxe2x80x9d Phe between Thr at position 91 and Gly at position 92 of the amino acid sequence of the CGTase from Thermoanaerobacter sp. ATCC 53627, cf. Table 1 (j).
Deletion of a proline at position 149 is indicated as P149*, and an insertion between position 147 and 148 where no amino acid residue is present, is indicated as *147aD for insertion of an aspartic acid in position 147a.
Multiple mutations are separated by slash marks (xe2x80x9c/xe2x80x9d), e.g. S145A/D147L, representing mutations in positions 145 and 147 substituting serine with alanine and aspartic acid with leucine, respectively.
If a substitution is made by mutation in e.g. a CGTase derived from a strain of Bacillus circulans, the product is designated e.g. xe2x80x9cB. circulans/S145Axe2x80x9d.
All positions referred to in this application by CGTase numbering refer to the CGTase numbers described above.